Protein Purification

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Pr otein Purification – Handbook

GE Healthcare

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Previous published Oct. 2001.

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Protein Purification

Handbook

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GST Gene Fusion System Handbook

18-1157-58

Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods

11-0012-69

2-D Electrophoresis

using immobilized pH gradients Principles and Methods 80-6429-60

Microcarrier Cell Culture Principles and Methods 18-1140-62

Challenging Protein Purification Handbook

28-9095-31

Recombinant Protein Purification Handbook Principles and Methods 18-1142-75

Protein Purification Handbook

18-1132-29 Gel Filtration Principles and Methods 18-1022-18

Affinity Chromatography Principles and Methods 18-1022-29

Antibody Purification Handbook

18-1037-46 Percoll

Methodology and Applications 18-1115-69

Ion Exchange Chromatography

& Chromatofocusing Principles and Methods 11-0004-21

Handbooks

from GE Healthcare

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Protein Purification

Handbook

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Introduction

The development of techniques and methods for protein purification has been an essential pre-requisite for many of the advancements made in biotechnology.

This handbook provides advice and examples for a smooth path to protein purification. Protein purification varies from simple one-step precipitation procedures to large scale validated production processes. Often more than one purification step is necessary to reach the desired purity. The key to successful and efficient protein purification is to select the most appropriate techniques,

optimise their performance to suit the requirements and combine them in a logical way to maximise yield and minimise the number of steps required.

Most purification schemes involve some form of chromatography. As a result chromatography has become an essential tool in every laboratory where protein purification is needed. Different chromatography techniques with different selectivities can form powerful combinations for the purification of any biomolecule.

The development of recombinant DNA techniques has revolutionised the production of proteins in large quantities. Recombinant proteins are often produced in forms which facilitate their subsequent chromatographic purification. However, this has not removed all challenges. Host contaminants are still present and problems related to solubility, structural integrity and biological activity can still exist.

Although there may appear to be a great number of parameters to consider, with a few simple guidelines and application of the Three Phase Purification Strategy the process can be planned and performed simply and easily, with only a basic knowledge of the details of chromatography techniques.

Advice codes:

general advice for any purification

advice for large scale purification

advice for micro scale purification

shortcuts

advice on media selection

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Chapter 1 Purification Strategies – a simple approach

Apply a systematic approach to development of a purification strategy.

The first step is to describe the basic scenario for the purification. General considerations answer questions such as: What is the intended use of the product?

What kind of starting material is available and how should it be handled? What are the purity issues in relation to the source material and intended use of the final product? What has to be removed? What must be removed completely? What will be the final scale of purification? If there is a need for scale-up, what consequences will this have on the chosen purification techniques? What are the economical constraints and what resources and equipment are available?

Most purification protocols require more than one step to achieve the desired level of product purity. This includes any conditioning steps necessary to transfer the product from one technique into conditions suitable to perform the next

technique. Each step in the process will cause some loss of product. For example, if a yield of 80% in each step is assumed, this will be reduced to only 20%

overall yield after 8 processing steps as shown in Figure 1. Consequently, to reach the targets for yield and purity with the minimum number of steps and the simplest possible design, it is not efficient to add one step to another until purity requirements have been fulfilled. Occasionally when a sample is readily available purity can be achieved by simply adding or repeating steps. However, experience shows that, even for the most challenging applications, high purity and yield can be achieved efficiently in fewer than four well-chosen and optimised purification steps. Techniques should be organised in a logical sequence to avoid the need for conditioning steps and the chromatographic techniques selected appropriately to use as few purification steps as possible.

Limit the number of steps in a purification procedure.

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Fig 1. Yields from multistep purifications.

Preparation

The need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to every purification. It is important to set objectives for purity, quantity and maintenance of biological activity and to define the economical and time framework for the work. All information concerning properties of the target protein and contaminants will help during purification development. Some simple experiments to characterise the sample and target molecule are an excellent invest- ment. Development of fast and reliable analytical assays is essential to

follow the progress of the purification and assess its effectiveness. Sample preparation and extraction procedures should be developed prior to the first chromatographic purification step.

With background information, assays and sample preparation procedures in place the Three Phase Purification Strategy can be considered.

Three Phase Purification Strategy

Imagine the purification has three phases Capture, Intermediate Purification and Polishing.

In the Three Phase Strategy specific objectives are assigned to each step within the process:

In the capture phase the objectives are to isolate, concentrate and stabilise the target product.

During the intermediate purification phase the objective is to remove most of the bulk impurities such as other proteins and nucleic acids, endotoxins and viruses.

In the polishing phase the objective is to achieve high purity by removing any remaining trace impurities or closely related substances.

The selection and optimum combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial to ensure fast method

development, a shorter time to pure product and good economy.

10 80 60 40 20 0

1 2 3 4 5 6 7 8 Number of steps

95% / step

90% / step 85% / step 80% / step 75% / step Yield (%)

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The final purification process should ideally consist of sample preparation, including extraction and clarification when required, followed by three major purification steps, as shown in Figure 2. The number of steps used will always depend upon the purity requirements and intended use for the protein.

Fig 2. Preparation and the Three Phase Purification Strategy.

Step

Purity

Capture

Intermediate purification

Polishing

Preparation, extraction, clarification

Achieve final high level purity

Remove bulk impurities Isolate, concentrate and stabilise

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Guidelines for Protein Purification

The guidelines for protein purification shown here can be applied to any purification process and are a suggestion as to how a systematic approach can be applied to the development of an effective purification strategy. As a reminder these guidelines will be highlighted where appropriate throughout the following chapters.

Define objectives

for purity, activity and quantity required of final product to avoid over or under developing a method

Define properties of target protein and critical impurities to simplify technique selection and optimisation

Develop analytical assays

for fast detection of protein activity/recovery and critical contaminants Minimise sample handling at every stage

to avoid lengthy procedures which risk losing activity/reducing recovery Minimise use of additives

additives may need to be removed in an extra purification step or may interfere with activity assays

Remove damaging contaminants early for example, proteases

Use a different technique at each step

to take advantage of sample characteristics which can be used for separation (size, charge, hydrophobicity, ligand specificity)

Minimise number of steps

extra steps reduce yield and increase time, combine steps logically

KEEP IT SIMPLE!

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Chapter 2 Preparation

Before You Start

The need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to any purification, from preparation of an enriched protein extract for biochemical characterisation to large scale production of a therapeutic recombinant protein. It is important to set objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time.

Purity requirements must take into consideration the nature of the source material, the intended use of the final product and any special safety issues. For example, it is important to differentiate between contaminants which must be removed and those which can be tolerated. Other factors can also influence the prioritisation of objectives. High yields are usually a key objective, but may be less crucial in cases where a sample is readily available or product is required only in small quantities. Extensive method development may be impossible without resources such as an ÄKTAdesign™ chromatography system. Similarly, time pressure combined with a slow assay turnaround will steer towards less extensive scouting and optimisation. All information concerning properties of the target protein and contaminants will help during purification development, allowing faster and easier technique selection and optimisation, and avoiding conditions which may inactivate the target protein.

Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess effectiveness (yield, biological activity, recovery).

Define objectives

Goal: To set minimum objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time.

Define purity requirements according to the final use of the product.

Purity requirement examples are shown below.

Extremely high > 99% Therapeutic use, in vivo studies

High 95- 99 % X-ray crystallography and most physico-chemical characterisation methods

Moderate < 95 % Antigen for antibody production N-terminal sequencing

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Identify 'key' contaminants

Identify the nature of possible remaining contaminants as soon as possible.

The statement that a protein is >95% pure (i.e. target protein constitutes 95% of total protein) is far from a guarantee that the purity is sufficient for an intended application. The same is true for the common statement "the protein was homogenous by Coomassie™ stained SDS-PAGE". Purity of 95% may be acceptable if the remaining 5% consists of harmless impurities. However, even minor impurities which may be biologically active could cause significant problems in both research and therapeutic applications. It is therefore important to differentiate between contaminants which must be removed completely and those which can be reduced to acceptable levels. Since different types of starting material will contain different contaminant profiles they will present different con- tamination problems.

It is better to over-purify than to under-purify.

Although the number of purification steps should be minimised, the quality of the end product should not be compromised. Subsequent results might be questioned if sample purity is low and contaminants are unknown.

Contaminants which degrade or inactivate the protein or interfere with analyses should be removed as early as possible.

The need to maintain biological activity must be considered at every stage during purification development. It is especially beneficial if proteases are

removed and target protein transferred into a friendly environment during the first step.

A downstream production process must achieve the required purity and recovery with complete safety and reliability, and within a given economic framework.

Economy is a very complex issue. In commercial production the time to mar- ket can override issues such as optimisation for recovery, capacity or speed.

Robustness and reliability are also of great concern since a batch failure can have major consequences.

Special safety issues may be involved in purification of biopharma- ceuticals, such as detection or removal of infectious agents, pyrogens, immunogenic contaminants and tumorigenic hazards.

It may be necessary to use analytical techniques targetted towards specific contaminants in order to demonstrate that they have been removed to acceptable levels.

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Define properties of target protein and critical impurities

Goal: To determine a 'stability window' for the target protein for easier selection and optimisation of techniques and to avoid protein inactivation during

purification.

Check target protein stability window for at least pH and ionic strength.

All information concerning the target protein and contaminant properties will help to guide the choice of separation techniques and experimental conditions for

purification. Database information for the target, or related proteins, may give size, isoelectric point (pI) and hydrophobicity or solubility data. Native one and two dimensional PAGE can indicate sample complexity and the properties of the target protein and major contaminants. Particularly important is a knowledge of the stability window of the protein so that irreversible inactivation is avoided. It is advisable to check the target protein stability window for at least pH and ionic strength. Table 1 shows how different target protein properties can affect a purification strategy.

Table 1. Protein properties and their effect on development of purification strategies.

Sample and target protein properties Influence on purification strategy Temperature stability Need to work rapidly at lowered temperature pH stability Selection of buffers for extraction and purification

Selection of conditions for ion exchange, affinity or reversed phase chromatography

Organic solvents stability Selection of conditions for reversed phase chromatography

Detergent requirement Consider effects on chromatographic steps and the need for detergent removal. Consider choice of detergent.

Salt (ionic strength) Selection of conditions for precipitation techniques, ion exchange and hydrophobic interaction chromatography

Co-factors for stability or activity Selection of additives, pH, salts, buffers

Protease sensitivity Need for fast removal of proteases or addition of inhibitors

Sensitivity to metal ions Need to add EDTA or EGTA to buffers Redox sensitivity Need to add reducing agents Molecular weight Selection of gel filtration media Charge properties Selection of ion exchange conditions Biospecific affinity Selection of ligand for affinity medium Post translational modifications Selection of group-specific affinity medium Hydrophobicity Selection of medium for hydrophobic interaction

chromatography

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Develop analytical assays

Goal: To follow the progress of a purification, to assess effectiveness (yield, biological activity, recovery) and to help during optimisation.

Select assays which are fast and reliable.

To progress efficiently during method development the effectiveness of each step should be assessed. The laboratory should have access to the following assays:

• A rapid, reliable assay for the target protein

• Purity determination

• Total protein determination

• Assays for impurities which must be removed

The importance of a reliable assay for the target protein cannot be over-

emphasised. When testing chromatographic fractions ensure that the buffers used for separation do not interfere with the assay. Purity of the target protein is most often estimated by SDS-PAGE, capillary electrophoresis, reversed phase

chromatography or mass spectrometry. Lowry or Bradford assays are used most frequently to determine the total protein.

The Bradford assay is particularly suited to samples where there is a high lipid content which may interfere with the Lowry assay.

For large scale protein purification the need to assay for target proteins and critical impurities is often essential. In practice, when a protein is purified for research purposes, it is too time consuming to identify and set up specific assays for harmful contaminants. A practical approach is to purify the protein to a certain level, and then perform SDS-PAGE after a storage period to check for protease cleavage. Suitable control experiments, included within assays for bio-activity, will help to indicate if impurities are interfering with results.

Sample Extraction and Clarification

Minimise sample handling Minimise use of additives

Remove damaging contaminants early

Definition: Primary isolation of target protein from source material.

Goal: Preparation of a clarified sample for further purification. Removal of particulate matter or other contaminants which are not compatible with chromatography.

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The need for sample preparation prior to the first chromatographic step is dependent upon sample type. In some situations samples may be taken directly to the first capture step. For example cell culture supernatant can be applied directly to a suitable chromatographic matrix such as Sepharose™ Fast Flow and may require only a minor adjustment of the pH or ionic strength. However, it is most often essential to perform some form of sample extraction and clarification procedure.

If sample extraction is required the chosen technique must be robust and suitable for all scales of purification likely to be used. It should be noted that a technique such as ammonium sulfate precipitation, commonly used in small scale, may be unsuitable for very large scale preparation. Choice of buffers and additives must be carefully considered if a purification is to be scaled up. In these cases

inexpensive buffers, such as acetate or citrate, are preferable to the more complex compositions used in the laboratory. It should also be noted that dialysis and other common methods used for adjustment of sample conditions are unsuitable for very large or very small samples.

For repeated purification, use an extraction and clarification technique that is robust and able to handle sample variability. This ensures a reproducible product for the next purification step despite variability in starting material.

Use additives only if essential for stabilisation of product or improved extraction. Select those which are easily removed. Additives may need to be removed in an extra purification step.

Use prepacked columns of Sephadex™ G-25 gel filtration media, for rapid sample clean-up at laboratory scale, as shown in Table 2.

Table 2. Prepacked columns for sample clean-up.

Sample volume Sample volume

Prepacked column loading per run recovery per run Code No.

HiPrep™ Desalting 26/10 2.5–15 ml 7.5–20 ml 17-5087-01

HiTrap™ Desalting 0.25–1.5 ml 1.0–2.0 ml 17-1408-01

Fast Desalting PC 3.2/10 0.05–0.2 ml 0.2–0.3 ml 17-0774-01

PD-10 Desalting 1.5–2.5 ml 2.5–3.5 ml 17-0851-01

Sephadex G-25 gel filtration media are used at laboratory and production scale for sample preparation and clarification of proteins >5000. Sample volumes of up to 30%, or in some cases, 40% of the total column volume are loaded. In a single step, the sample is desalted, exchanged into a new buffer, and low molecular weight materials are removed. The high volume capacity, relative insensitivity to sample concentration, and speed of this step enable very large sample volumes to be processed rapidly and efficiently. Using a high sample volume load results in a separation with minimal sample dilution (approximately 1:1.4). Chapter 8 contains further details on sample storage, extraction and clarification

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Sephadex G-25 is also used for sample conditioning, e.g. rapid adjustment of pH, buffer exchange and desalting between purification steps.

Media for consideration:

Sephadex G 25 gel filtration

For fast group separations between high and low molecular weight substances Typical flow velocity 60 cm/h (Sephadex G-25 Superfine, Sephadex G-25 Fine), 150 cm/h (Sephadex G-25 Medium).

Combine Sample Clean-up and Capture in a single step

If large sample volumes will be handled or the method scaled-up in the future, consider using STREAMLINE™ expanded bed adsorption. This technique is particularly suited for large scale recombinant protein and monoclonal antibody purification. The crude sample containing particles can be applied to the

expanded bed without filtration or centrifugation. STREAMLINE adsorbents are specially designed for use in STREAMLINE columns. Together they enable the high flow rates needed for high productivity in industrial applications of fluidised beds. The technique requires no sample clean up and so combines sample

preparation and capture in a single step. Crude sample is applied to an expanded bed of STREAMLINE media. Target proteins are captured whilst cell debris, cells, particulate matter, whole cells, and contaminants pass through. Flow is reversed and the target proteins are desorbed in the elution buffer.

STREAMLINE (IEX, AC, HIC)

For sample clean-up and capture direct from crude sample.

STREAMLINE adsorbents are designed to handle feed directly from both fermentation homogenate and crude feedstock from cell culture/fermentation at flow velocities of 200 - 500 cm/h, according to type and application.

Note: cm/h: flow velocity (linear flow rate) = volumetric flow rate/cross sectional area of column.

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Chapter 3 Three Phase Purification Strategy

Principles

With background information, assays, and sample preparation and extraction procedures in place the Three Phase Purification Strategy can be applied (Figure 3). This strategy is used as an aid to the development of purification processes for therapeutic proteins in the pharmaceutical industry and is equally efficient as an aid when developing purification schemes in the research

laboratory.

Fig 3. Preparation and the Three Phase Purification Strategy.

Assign a specific objective to each step within the purification process.

In the Three Phase Strategy a specific objective is assigned to each step. The purification problem associated with a particular step will depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process, i.e. at the beginning for isolation of product from crude sample, in the middle for further purification of partially purified sample, or at the end for final clean up of an almost pure product.

The Three Phase Strategy ensures faster method development, a shorter time to pure product and good economy.

In the capture phase the objectives are to isolate, concentrate and stabilise the target product. The product should be concentrated and transferred to an environment which will conserve potency/activity. At best, significant removal of

Step

Purity

Capture

Intermediate purification

Polishing

Preparation, extraction, clarification

Achieve final high level purity

Remove bulk impurities Isolate, concentrate and stabilise

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other critical contaminants can also be achieved.

During the intermediate purification phase the objectives are to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins and viruses.

In the polishing phase most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity.

It should be noted that this Three Phase Strategy does not mean that all strategies must have three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result, or the purity of the starting material may be so high that only a polishing step is needed. For purification of therapeutic proteins a fourth or fifth purification step may be required to fulfil the highest purity and safety demands.

The optimum selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification process.

Selection and Combination of Purification Techniques

Every technique offers a balance between resolution, capacity, speed and recovery.

Minimise sample handling Minimise number of steps

Use different techniques at each step

Goal: Fastest route to a product of required purity.

For any chromatographic separation each different technique will offer different performance with respect to recovery, resolution, speed and capacity. A technique can be optimised to focus on one of these parameters, for example resolution, or to achieve the best balance between two parameters, such as speed and capacity.

A separation optimised for one of these parameters will produce results quite different in appearance from those produced using the same technique, but focussed on an alternative parameter. See, for example, the results shown on page 49 where ion exchange is used for a capture and for a polishing step.

Speed

Recovery

Capacity Resolution

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Select a technique to meet the objectives for the purification step.

Capacity, in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample which can be loaded may be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein.

Speed is of the highest importance at the beginning of a purification where contaminants such as proteases must be removed as quickly as possible.

Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and unfavourable conditions on the column.

Resolution is achieved by the selectivity of the technique and the efficiency of the chromatographic matrix to produce narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties.

Every technique offers a balance between resolution, speed, capacity and recovery and should be selected to meet the objectives for each purification step. In

general, optimisation of any one of these four parameters can only be achieved at the expense of the others and a purification step will be a compromise. The importance of each parameter will vary depending on whether a purification step is used for capture, intermediate purification or polishing. This will steer the optimisation of the critical parameters, as well as the selection of the most suitable media for the step.

Proteins are purified using chromatographic purification techniques which separate according to differences in specific properties, as shown in Table 3.

Table 3. Protein properties used during purification.

Protein property Technique

Charge Ion exchange (IEX)

Size Gel filtration (GF)

Hydrophobicity Hydrophobic interaction (HIC),

Reversed phase (RPC) Biorecognition (ligand specificity) Affinity (AC)

Charge, ligand specificity or hydrophobicity Expanded bed adsorption (EBA) follows the principles of AC, IEX or HIC

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Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step.

Minimise sample handling between purification steps by combining techniques to avoid the need for sample conditioning.

A guide to the suitability of each purification technique for the stages in the Three Phase Purification Strategy is shown in Table 4.

Table 4. Suitability of purification techniques for the Three Phase Purification Strategy

Avoid additional sample conditioning steps.

The product should be eluted from the first column in conditions suitable for the start conditions of the next column.

The start conditions and end conditions for the techniques are shown in Table 4.

For example, if the sample has a low ionic strength it can be applied to an IEX column. After elution from IEX the sample will usually be in a high ionic strength buffer and can be applied to a HIC column (if necessary the pH can be adjusted and further salt can be added). In contrast, if sample is eluted from a HIC column, it is likely to be in high salt and will require dilution or a buffer exchange step in order to further decrease the ionic strength to a level suitable for IEX. Thus it is more straightforward to go from IEX to HIC than vice-versa.

Ammonium sulfate precipitation is a common sample clarification and concentration step at laboratory scale and in this situation HIC (which requires high salt to enhance binding to the media) is ideal as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a Sephadex G-25 desalting column will prepare it for the next IEX or AC step.

Technique Main features Capture Intermediate Polish Sample Start Sample End condition condition

IEX high resolution low ionic strength high ionic

high capacity sample volume strength or

high speed not limiting pH change

concentrated

HIC good resolution high ionic strength low ionic

good capacity sample volume strength

high speed not limiting concentrated

AC high resolution specific binding specific

high capacity conditions elution

high speed sample volume conditions

not limiting concentrated

GF high resolution limited sample buffer

using Superdex™ volume (<5% total exchanged (if

column volume) required) and flow rate diluted range

RPC high resolution requires organic in organic

solvents solvent, risk loss of biological activity concentrated

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GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation (gel filtration is a non-binding technique with limited volume capacity and unaffected by buffer conditions).

Selection of the final strategy will always depend upon specific sample properties and the required level of purification. Logical combinations of techniques are shown in Figure 4.

Fig 4. Logical combinations of chromatographic steps.

For any capture step, select the technique showing the strongest binding to the target protein while binding as few of the contaminants as possible, i.e. the technique with the highest selectivity and/or capacity for the protein of interest.

Crude sample or sample in high salt concentration

GF GF GF

desalt modedesalt mode desalt mode

AC IEX HIC IEX

no need to desalt

IEX HIC

GF GF GF GF

or or

RPC RPC

Proteins with low solubility

SDS SDS Solubilizing agents

extraction extraction (urea, ethylene glycol non-ionic detergents)

GF HIC HIC

(in non-ionic detergent)

GF GF

Chap 1. Fig 4–6

Clear or very dilute samples

AC IEX IEX Precipitation

(e.g. in high ionic strength)

HIC Resolubilise

GF GF GF Treat as for

or or sample in high salt

RPC RPC concentration

Capture

Intermediate Purification Polishing

Sample clarification

Capture

Intermediate Purification Polishing

Crude sample or sample in high salt concentration

GF GF GF

desalt modedesalt mode desalt mode

AC IEX HIC IEX

dilution may be needed

IEX HIC

GF GF GF GF

Sample clarification*

Capture

Intermediate Purification

Polish

* Alternatively samples can be filtered and, if required, their ionic strength can be reduced by dilution.

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A sample is purified using a combination of techniques and alternative

selectivities. For example, in an IEX-HIC-GF Three Phase Strategy the capture step selects according to differences in charge (IEX), the intermediate purification step according to differences in hydrophobicity (HIC) and the final polishing step according to differences in size (GF). Figure 5 shows a standard Three Phase strategy purification.

Fig 5. A standard purification protocol.

If nothing is known about the target protein use IEX-HIC-GF.

This combination of techniques can be regarded as a standard protocol.

Consider the use of both anion and cation exchange chromatography to give different selectivities within the same purification strategy.

IEX is a technique which offers different selectivities using either anion or cation exchangers. The pH of the separation can be modified to alter the charge characteristics of the sample components. It is therefore possible to use IEX more than once in a purification strategy, for capture, intermediate purification or polishing. IEX can be used effectively both for rapid separation in low resolution mode during capture, and in high resolution mode during polishing in the same purification scheme. Figure 6 shows an example for the purification of cellulase in which advantage is taken of the different selectivities of anion and cation exchange to create a simple two step process.

Capture by IEX Basic proteins

STREAMLINE SP or SP Sepharose XL

Suggested binding buffer: 20 mM sodium phosphate, pH 7 Suggested elution buffer: Binding buffer + 0.5 M NaCl Acidic proteins

STREAMLINE DEAE or Q Sepharose XL Suggested binding buffer: 50 mM Tris.HCl, pH 8 Suggested elution buffer: Binding buffer + 0.5 M NaCl

Intermediate purification by HIC Phenyl Sepharose 6 Fast Flow (high sub)

Suggested binding buﬀer: 50 mM sodium phosphate, pH 7 + 1.5 M ammonium sulfate Suggested elution buﬀer: 50 mM sodium phosphate, pH 7

Polishing by GF

Superdex 75 prep grade or Superdex 200 prep grade Suggested buﬀer: as required by subsequent use

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Fig 6. Two step purification of a cellulase.

Consider RPC for a polishing step provided that the target protein can withstand the run conditions.

Reversed phase chromatography (RPC) separates proteins and peptides on the basis of hydrophobicity. RPC is a high selectivity (high resolution) technique, requiring the use of organic solvents. The technique is widely used for purity check analyses when recovery of activity and tertiary structure are not essential.

Since many proteins are denatured by organic solvents, the technique is not generally recommended for protein purification where recovery of activity and return to a correct tertiary structure may be compromised. However, in the polishing phase, when the majority of protein impurities have been removed, RPC can be excellent, particularly for small target proteins which are often not denatured by organic solvents.

If a purification is not intended for scale up (i.e. only milligram quantities of product are needed), use high performance, prepacked media such as Sepharose High Performance (IEX, HIC), SOURCE™ (IEX, HIC), MonoBeads™ (IEX), or Superdex (GF) for all steps.

Recommended media for a standard protocol

Purification step Media Quantity Code No.

Capture STREAMLINE SP 300 ml 17-0993-01

Capture STREAMLINE DEAE 300 ml 17-0994-01

Capture HiPrep 16/10 SP XL 1 column 17-5093-01

Capture HiPrep 16/10 Q XL 1 column 17-5092-01

Intermediate purification HiPrep 16/10 Phenyl FF (high sub) 1 column 17-5095-01 Polishing HiLoad™ 16/60 Superdex 75 prep grade 1 column 17-1068-01 Polishing HiLoad 16/60 Superdex 200 prep grade 1 column 17-1069-01 Sample clarification/conditioning Prepacked PD-10 Column 30 columns 17-0851-01 Sample clarification/conditioning HiTrap Desalting 5 columns 17-1408-01

A_280nm 0.5

0 10 20 30

Time (min) (3)

A_280nm 0.5

0 10 20

Time (min) (3)

Sample:

Column:

Flow rate:

Buffer A:

Buffer B.

Gradient:

Peak 3 from step 1 Mono S™ HR 5/5 1.0 ml/min 20 mM acetate, pH 3.6 A + 0.2 M NaCl 0-100% B in 26 min Sample:

Column:

Flow:

Buffer A:

Buffer B.

Gradient:

500 µl of Trichoderma reesei crude cellulases in buffer A, 2.5 mg

Mono Q™ HR 5/5 1.0 ml/min 20 mM Tris-HCl, pH 7.6 A + 0.5 M NaCl

0% B for 4 min, 0-40% in 21 min, 40-100% B in 15 min

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Sample Conditioning

Although additional sample handling between purification steps should be avoided, it may be necessary to adjust the buffer conditions of an eluted product (pH, ionic strength and/or buffering ions) to ensure compatibility with the following purification technique.

Sephadex G-25 is an ideal media for rapid desalting and pH adjustment by buffer exchange between purification steps. Sample volumes of up to 30%, or in some cases 40%, of the total column volume are loaded. In a single step, the sample is desalted, exchanged into a new buffer, and low molecular weight materials are removed. Figure 7 shows a typical desalt/buffer exchange separation. The high volume capacity and speed of this step enable very large sample volumes to be processed rapidly and efficiently. The high sample volume load results in a separation with minimal sample dilution. Sephadex G-25 is also used for rapid sample clean-up at laboratory scale.

Fig 7. Buffer exchange of mouse plasma on HiPrep 26/10 Desalting.

Use prepacked columns of Sephadex G-25 for rapid sample conditioning at laboratory scale, as shown in Table 5.

Table 5. Prepacked columns for rapid desalting and buffer exchange.

Prepacked column Sample volume Sample volume Code No.

loading per run recovery per run

HiPrep Desalting 26/10 2.5 -15 ml 7.5 - 20 ml 17-5087-01

HiTrap Desalting 0.25 - 1.5 ml 1.0 - 2.0 ml 17-1408-01

Fast Desalting PC 3.2/10 0.05 - 0.2 ml 0.2 - 0.3 ml 17-0774-01

PD-10 Desalting 1.5 - 2.5 ml 2.5 - 3.5 ml 17-0851-01

0.0 1.0 2.0

0.00 0.05 0.10 0.15 0.20 0.25

A_{280 nm}

A280

5.0 10.0

mS/cm

Time (min) Conductivity

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Dilution can be used as an alternative to desalting before application to an ion exchange column.

Sephadex G-25 Gel filtration

For fast group separations between high and low molecular weight substances.

Typical flow velocities 60 cm/h (Sephadex G-25 Superfine, Sephadex G-25 Fine), 150 cm/h (Sephadex G-25 Medium).

In the following chapters Capture, Intermediate Purification and Polishing are discussed in more detail.

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Chapter 4 Capture

Remove damaging contaminants early

Definition: Initial purification of the target molecule from crude or clarified source material.

Goals: Rapid isolation, stabilisation and concentration.

Use a high capacity, concentrating technique to reduce sample volume, to enable faster purification and to allow the use of smaller columns.

Focus on robustness and simplicity in the first purification step. Do not try to solve all problems in one step when handling crude material.

In the capture phase, the objective is to isolate, concentrate and stabilise the target product efficiently by optimising speed and capacity. The product is concentrated and transferred to an environment which will conserve activity. Capture is often a group separation using a step elution on ion exchange or affinity chromatography.

Ideally, removal of critical contaminants is also achieved. It is sometimes possible to achieve a high level of purification if a highly selective affinity media is used.

Binding capacity for the protein in the presence of the impurities will be one of the most critical parameters to optimise and reduce the scale of work. For example, when ion exchange chromatography is used as a capture step, the goal is to adsorb the target protein quickly from the crude sample and isolate it from critical contaminants such as proteases and glycosidases. Conditions are selected to avoid binding of contaminants so that the capacity for the target protein is maximised.

High speed may be required to reduce sample application time, particularly if proteolysis or other destructive effects threaten the integrity of the target protein.

Transfer to a step elution during method development to increase speed and capacity of the capture step.

Speed

Recovery

Capacity Resolution

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The most common technique for a capture step is ion exchange chromatography (IEX) which has high binding capacity. IEX media are resistant to harsh cleaning conditions which may be needed after purification of crude samples. Typically proteins are eluted from an IEX column using a salt gradient. However, during method development, a transfer to a step elution will give a simple, robust separation with a shorter run time and decreased buffer consumption. It is often possible to use high sample loadings since the focus is not on resolution (high sample loadings will decrease resolution). High speed and capacity and low buffer consumption are particularly advantageous for large scale purification, as shown in Figure 8.

Fig. 8. Examples of capture steps.

100

%B

150

15 150 Volume (I)

50 100

0 0 1.0 2.0 3.0

A_{280 nm}

EGF

A_{280 nm} Conductivity

0 5.0 10.0 15.0 20.0 Volume (l)

A 2.0

1.5

1.0

0.5

0.0

0 200 400 600 Volume (m)

280 nm

Column: INdEX™ 70 (70 mm i.d.) Adsorbent: Q Sepharose XL, 385 mL bed volume Sample: Recombinant α-amylase produced in E. coli, homogenized, 2.2 L diluted in distilled water to 15.4 L, 7.2 mS/cm, 10 mM CaCl2, centrifuged Buffer A: 20 mM Tris-HCl, pH 8, 10 mM CaCl2 Buffer B: 20 mM Tris-HCl, pH 8, 1 M NaCl,

10 mM CaCl2 Flow: 300 cm/h, 12 L/h Gradient: 20 bed volumes 0-1 M NaCl Eluate: 1.48 L, 3.8 bed volumes Spec. act.

α-amylase: 6420 U/L

Column: rProtein A Sepharose Fast Flow, XK 16/20, bed height 4.8 cm (9.6 mL) Sample: clarified cell culture containing IgG2a Sample volume: 600 mL containing 87.6 mg IgG2a Starting buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 20 mM sodium citrate, pH 4.0 Flow: 5 mL/min (150 cm/h) Column: BPG™ 300/500 packed with

Phenyl Sepharose 6 Fast Flow (high sub) Sample: EGF in yeast supernatant

ammonium sulfate added to 0.5 M Sample load: 80 L containing 2.56 g EGF Starting buffer: 20 mM sodium phosphate, pH 7.0 + 0.5 M

ammonium sulfate

Elution buffer: 20 mM sodium phosphate, pH 7.0 Flow loading: 210 L/h, 300 cm/h

a

b

c

a) Purification of recombinant epidemal growth factor (EGF) - capture step.

b) Pilot scale purification of recombinant α-amylase from E. coli - capture step.

c) Purification of IgG_2a from clarified cell culture - capture step.

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For large scale capture, throughput will often be the focus during method development. It is important to consider all aspects: sample extraction and clarification, sample loading capacity, flow rate during equilibration, binding, washing, elution and cleaning, and the need for cleaning-in-place procedures.

In principle, a capture step is designed to maximise capacity and/or speed at the expense of some resolution. However, there is usually significant resolution and purification from molecules which have significant physicochemical differences compared to the target protein. Recovery will be of concern in any preparative situation, especially for production of a high value product, and it is important to assay for recovery during optimisation of the capture step. Examples of capture steps are shown on page 30.

Media for capture steps should offer high speed and high capacity.

Sepharose XL (IEX)

For capture steps handling crude mixtures at laboratory and process scale. Fast removal and a combination of high capacity and good resolution at high flow rates are the main characteristics. Recommended flow velocity is

100-500 cm/h.

Particle size: 90 µm. Available in prepacked columns and as bulk media.

Sepharose Big Beads (IEX)

For capture steps handling viscous samples or very large sample volumes.

Sepharose Big Beads are for the capture step in processes where high sample viscosity precludes the use of ion exchange media with smaller bead sizes.

Recommended flow velocity is up to 300 cm/h. This medium should be chosen when fast adsorption is required and resolution is of less importance. The flow characteristics of Big Beads may also be useful when processing very large volumes under conditions requiring an extremely high volumetric throughput. Flow

velocities in these situations can exceed 1000 cm/h.

Particle size: 200 µm. Available as bulk media.

STREAMLINE (IEX, AC, HIC)

For sample clean-up and capture direct from crude sample.

STREAMLINE adsorbents are designed to handle feed directly from both fermentation homogenate and crude feedstock from cell culture/fermentation at flow velocities of 200 - 500 cm/h, according to type and application.

Particle size: 200 µm. Available as bulk media.

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Other media for consideration:

Sepharose Fast Flow (IEX, HIC)

These media offer the widest range of selectivities and an excellent alternative for purification of crude samples at any scale. They offer a fast separation combined with good resolution. Recommended flow velocity is 100-300 cm/h.

Particle size: 90 µm. Available in prepacked columns and as bulk media.

If a purification is not intended for scale up (i.e. milligram quantities of product are needed), use high performance media such as Sepharose High Performance (IEX, HIC) or MonoBeads (IEX), or SOURCE (IEX, HIC).

All these media are available in prepacked columns.

For microscale purification use MonoBeads or MiniBeads™ (IEX), Phenyl Superose™ (HIC) or NHS-activated Superose (AC) columns.

For 'one time' purification or with a readily available sample, sacrifice yield for purity by taking a narrow cut from a chromatographic peak during the first purification step.

Use HiTrap Ion Exchange and HiTrap HIC Test Kits for media screening and simple method optimisation.

If the starting material is reasonably clean, a single step purification on highest resolution MonoBeads (IEX) may be sufficient to achieve required purity at laboratory scale.

If a biospecific ligand is available, consider using affinity chromatography as the capture step. If the media is to be used routinely, ensure that any contaminants from the crude sample can be removed by column regeneration procedures which do not damage the affinity ligand. AC will give a highly selective capture step to improve resolution from contaminants, but speed may need to be reduced to maintain a high binding capacity.

If the starting material is reasonably clean a single step purification on a prepacked HiTrap affinity column may be sufficient to achieve required purity at the milligram scale, as shown in Figure 9. HiTrap affinity columns are available in a wide range of selectivities (see Table 6, page 34).

If the starting material is concentrated, has a low volume and there is no intention to scale up, Superdex gel filtration media can offer a mild first step, requiring little or no optimisation. Conversely, gel filtration is not

suitable in a typical capture step where the sample volume is large or will be scaled up.

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Fig 9. HiTrap Chelating column used to purify histidine-tagged glutathione-S-transferase from cytoplasmic extract.

280 nm

A A_{405 nm}

Column: HiTrap Chelating, 1 ml

Sample: 5 ml cytoplasmic extract containing histidine-tagged glutathione-S-transferase

Binding buffer: 20 mM phosphate buffer, 0.5 M NaCl, 20 mM imidazole, pH 7.4 Elution buffer: 20 mM phosphate buffer, 0.5 M NaCl,

500 mM imidazole, pH 7.4 Flow: 2 mL/min (312 cm/h)

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Application

Isolation of immunoglobulins IgG classes, fragments and subclasses IgG classes, fragments and subclasses

IgG classes, fragments and subclasses including human IgG₃ strong affinity for monoclonal mouse IgG₁ and rat IgG Monoclonal and poly- clonal IgG from ascites fluid, serum and cell culture supernatant

Mouse recombinant Single chain antibody Fragment variable (ScFv) produced in E. coli

IgY antibodies from egg yolk

IgM

HiTrap column

HiTrap rProtein A

HiTrap Protein A

HiTrap Protein G

MAbTrap™ GII

RPAS Purification Module

HiTrap IgY Purification HiTrap IgM Purification

Code No.

17-5079-01 17-5080-01 17-5029-02

17-0402-01 17-0402-03 17-0403-01 17-0404-01 17-0404-03 17-0405-01

17-1128-01

17-1362-01

17-5111-01

17-5110-01

Quantity/

components 5 x 1 ml 1 x 5 ml 2 x 1 ml

5 x 1 ml 2 x 1 ml 1 x 5 ml 5 x 1 ml 2 x 1 ml 1 x 5 ml

HiTrap Protein G column (1 ml), accessories, pre-made buffers for 10 purifications

HiTrap Anti-E column, accessories, pre-made buffers for 20 purifications

1 x 5 ml

5 x 1 ml

Approximate binding capa- city per ml gel human IgG 50 mg/ml

human IgG 20 mg/ml

human IgG 25 mg/ml

as above

0.17 mg ScFv/5 ml

IgY 20 mg/ml

IgM 5 mg/ml Table 6. Recommended HiTrap affinity columns for laboratory scale separation.

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Application

Group Specific Media Various Nucleotide- requiring enzymes, coagulation factors, DNA binding proteins, α2-macroglobulin Proteins and peptides with exposed amino acids: His (Cys, Trp) e.g. α2-macroglobulin and interferon

Histidine-tagged fusion proteins

Biotin and biotinylated substances

Coagulation factors, lipoprotein lipases, steroid receptors, hormones, DNA binding proteins, interferon, protein synthesis factors Matrix for preparation of affinity media Coupling of primary amines

HiTrap column

HiTrap Blue

HiTrap Chelating

HisTrap™

HiTrap Streptavidin HiTrap Heparin

HiTrap NHS-activated

Code No.

17-0412-01 17-0413-01

17-0408-01 17-0409-01

17-1880-01

17-5112-01

17-0406-01 17-0407-01

17-0716-01 17-0717-01

Quantity/

components 5 x 1 ml 1 x 5 ml

5 x 1 ml 1 x 5 ml

HiTrap Chelating column (1 ml), accessories, pre-made buffers

5 x 1 ml

5 x 1 ml 1 x 5 ml

Approximate binding capa- city per ml gel HSA 20 mg/ml

histidine-tagged protein (27.6 kD) 12 mg /ml

as above

biotinylated BSA 6 mg/ml ATIII (bovine) 3 mg/ml

ligand specific

Recommended separation conditions

All HiTrap columns are supplied with a detailed protocol to ensure optimum results Maximum flow rates: HiTrap 1 ml column: up to 4 ml/min

HiTrap 5 ml column: up to 20 ml/min

Protein Purification

Pr otein Purification – Handbook

GE Healthcare

Protein Purification

Handbook

Handbooks

from GE Healthcare

Protein Purification

Handbook

Contents

Introduction

Chapter 1

Purification Strategies – a simple approach

Preparation

Three Phase Purification Strategy

Guidelines for Protein Purification

KEEP IT SIMPLE!

Chapter 2

Preparation

Before You Start

Define objectives

Define properties of target protein and critical impurities

Develop analytical assays

Sample Extraction and Clarification

Chapter 3

Three Phase Purification Strategy

Principles

Selection and Combination of Purification Techniques

Sample Conditioning

Chapter 4

Capture

Sepharose XL (IEX)

Sepharose Big Beads (IEX)

STREAMLINE (IEX, AC, HIC)

Sepharose Fast Flow (IEX, HIC)